Mitogenic activity of purified capsular polysaccharide A from Bacteroides fragilis: differential stimulatory effect on mouse and rat lymphocytes in vitro

Bacteroides fragilis, a Gram-negative colonic bacterium, induces the formation of abscesses associated with intra-abdominal sepsis in humans. The singular ability of this organism to modulate abscess formation in experimental rodent models resides in the structurally distinct and ionically charged capsular polysaccharides A (PS A) and B (PS B). The regulation of abscess formation in animals is dependent on T lymphocytes. However, the manner in which PS A interacts with T cells remains unknown. We therefore tested the T cell stimulatory capacity of purified PS A on mouse and rat lymphocytes in cellular proliferation assays and found that the PS A molecule possesses mitogenic characteristics distinguishable from those of the polyclonal B cell activator LPS, the T cell mitogen Con A, and staphylococcal enterotoxin A superantigen. Further, PS A stimulated proliferation of normal mouse and rat lymphocytes differentially. Mouse B cells responded to PS A in a fashion that did not require exogenous APC function, while rat T lymphocyte responses to PS A required APC function derived from autologous or xenogenic feeder cells. Cellular depletion experiments showed that the CD4+ subset of rat spleen cells was the primary responder cell type to PS A in vitro. The differential stimulatory effects of PS A on mouse and rat lymphocytes may reflect its ability to stimulate different lymphocyte subsets in vivo through the activities of receptor/counter-receptor pairs present on responder lymphocytes and cognate APC.     

Reovirus antibodies among animals in Taiwan

Serum specimens for reovirus antibody survey were collected from 60 pigs, 50 buffalos, 30 rabbits, 30 guinea pigs, 20 albino rats, 60 Swiss mice, 15 Taiwan monkeys, 14 goats and 12 dogs. Serum antibodies were measured by the hemagglutination inhibition (HI) test. HI antibodies to reovirus were found in a high percentage of pigs, albino rats, Swiss mice, dogs and goats, and less frequently in rabbits, guinea pigs, Taiwan monkeys and buffalos.     

In vitro stimulation of lymphocytes by neutral proteinases from human polymorphonuclear leukocyte granules

Two neutral proteinases from human polymorphonuclear leukocytes (PMN), an elastase and the chymotrypsin-like cathepsin G, were purified, and their actions on lymphocytes in culture were studied. Both PMN proteinases stimulate lymphocytes from human peripheral blood and from mouse spleen in vitro, but do not affect thymic cells from either normal or hydrocortisone-treated mice. In stimulated mouse spleen cell cultures, most of the developing blast cells bear surface immunoglobulins, and subsequently appear to engage in antibody synthesis. In their stimulatory action, the two PMN proteinases thus resemble the classic B-cell mitogen LPS and neutral pancreatic proteinases such as trypsin, chymotrypsin, and elastase. The effects of proteinase inhibitors indicate that lymphocyte stimulation is dependent on the proteolytic activity of the enzymes. This work suggests that PMN proteinases, which are released at sites of inflammation, may modulate the function of lymphocytes.     

Effects of vitamin B6 deficiency on cytokine levels and lymphocytes in mice

The effects of vitamin B6 (B6) deficiency on cytokine levels and proportions of lymphocyte subsets in BALB/c mice were investigated. The proportion of lymphocytes from the thymus and spleen of mice given no B6, that were CD4+ CD8- T cells, was larger than in mice given B6, and the ratio of CD8+ to CD4+ T cells in the thymus of mice given no B6 was lower. The concentrations of interleukin-5 and -10 in spleen cells stimulated in vitro with concanavalin A were significantly higher in the mice with B6 deficiency, as was their plasma corticosterone concentrations. These results suggested that B6 is necessary to maintain cytokine levels and lymphoid function in the thymus and spleen of mice.     

Characterization of the mitogenic response of murine CD5+ and conventional B lymphocytes to lipopolysaccharide

The nature of the response of conventional and CD5+ B cells to stimulation in vitro with optimal mitogenic concentrations of LPS was examined to elucidate the contributions of these B cell subsets in polyclonal B lymphocyte responses. Stimulation of murine splenic lymphocytes with LPS resulted in an increase in total biomass, peaking at 72 h of culture. The viability of the cultures remained high (> 90%) until 48 h of culture. A combination of trypan blue and 7-aminoactinomycin D (7AAD) exclusion in conjunction with PE-anti-CD5 and FITC-anti-B220 enabled more detailed analysis of the cultures. The total number of conventional B cells, viable and non-viable, increased until 48 h of culture and then decreased when stimulated with LPS, while CD5+ B cells increased over the culture period. The numbers of conventional B cells in the control cultures decreased, but the CD5+ B cell numbers remained stable. An examination of the modes of death of the B cell subsets using 7AAD showed that unstimulated conventional B cells were apoptotic rather than degenerate but, following stimulation with LPS, apoptotic and degenerate cells were found. Apoptotic and degenerate CD5+ B cells were found in both stimulated and unstimulated cultures, but the percentage of these apoptotic and degenerate cells was increased significantly only at 72 h and 96 h of culture in stimulated cultures compared with 24 h onwards in the control cultures. Morphological analysis and gel electrophoretic studies of extracted DNA reflected these findings. It was also found that the increase in the number and percentage of non-viable cells in the cultures was not equal to the decrease in the number and percentage of viable cells. Activation of B cells was examined using expression of B7-1 (CD80) as a marker. When stimulated with LPS a greater proportion of conventional B cells expressed B7-1 after 24 h of culture than in the control cultures; however, only at 72 h and 96 h of culture was the proportion of CD5+ B cells expressing B7-1 significantly higher than in the control cultures. These results show that conventional B cells are stimulated to proliferate and to become activated by LPS and that death is apoptotic rather than degenerate or necrotic. CD5+ B cells were also shown to be stimulated by LPS; they became activated and death was delayed. The data suggest that in addition to the proliferative role, LPS acts to delay death and to activate conventional and CD5+ B cells.     

Overload syndromes of the peritalar region

BACKGROUND: The purpose of this study was to investigate lymphocyte adhesion to Kupffer cells as a component of an immune-mediated mechanism for halothane hepatitis. METHODS: Kupffer cells were isolated from guinea pigs exposed to 1.0% halothane/40% oxygen and cultured with various synthetic antigens (trifluoroacetyl-protein adducts or hepatocyte homogenate from halothane-exposed animals). Latex beads were also added to Kupffer cell cultures to determine if activation of these macrophages would result in an increased cellular adhesion. Lymphocytes which had been surfaced-labeled with biotin were added to treated Kupffer cells, and lymphocyte adhesion was determined using a streptavidin-peroxidase reagent for colorimetric detection. RESULTS: Trifluoroacetyl-lysine, trifluoroacetyl-rabbit albumin or guinea pig albumin did not induce lymphocyte adhesion. Latex beads also had no effect on cellular adhesion. A noticeable increase in lymphocyte adhesion to Kupffer cells previously treated with either trifluoroacetyl-guinea pig albumin or hepatocyte homogenate was observed. Stimulation of lymphocytes with phorbol myristate acetate did not have an effect on adhesion. Addition ofantimajor histocompatibility complex II antibody had a significant inhibitory effect on lymphocyte adhesion to Kupffer cells treated with trifluoroacetyl-guinea pig albumin or homogenate. CONCLUSION: These results demonstrate that the halothane trifluoroacetyl-guinea pig albumin antigen and hepatocyte homogenate enhances the adhesion of lymphocytes to cultured Kupffer cells and that this interaction involves major histocompatibility complex II expression on stimulated Kupffer cells. The interaction between Kupffer cells which present specific trifluoroacetyl-antigens and lymphocytes from halothane-exposed animals may play an important role in the pathogenesis of halothane hepatitis.     

Effects of somatostatin and captopril on glomerular prostaglandin E2 production in normal and diabetic rats

The macular mutant mouse is a murine model of the Menkes' kinky hair disease, characterized by a copper deficiency in serum. The immune response of its hemizygote (ml/y) was examined, herein. Ml/y mice which were not treated with Cu were atrophy of lymphoid tissues on day 14. However, kidney, brain, heart and lung weights were higher in ml/y mice without Cu treatment than in normal (+/y) mice. When compared to cells from +/y mice, spleen cells from ml/y mice exhibited similar proliferation-curves stimulated by Con A or LPS. Lymph node cells from ml/y mice showed a significantly decreased mixed lymphocyte reaction (MLR) response when stimulated by spleen cells from Balb/c mice. Spleen cells from ml/y mice demonstrated similar stimulation against lymph node cells from Balb/c mice. Antibody production against sheep red blood cells (SRBC) in vivo, a T cell dependent response, was suppressed in ml/y mice. By contrast, the antibody production against dinitrophenyl-ficoll, a T cell independent response was similar in +/y and ml/y. The antibody production against SRBC in vitro was also suppressed in ml/y mice. However, when the T cell-enriched fraction of ml/y mouse spleen cells was replaced by the T cell-enriched fraction of +/y mouse spleen cells, the antibody production against SRBC was recovered. The percentage of Ly-5-positive cells (B cell) from ml/y mice was greater than those from +/y mice. The percentage of Thy-1.2-positive cells (T cell) was decreased, and the decrease was most prominent with the L3/T4-positive T cell (helper T cell) subset.     

Modulation of T cell and monocyte function in the spleen following infection of pigs with African swine fever virus

Infection of pigs with many strains of African Swine Fever Virus (ASFV) has been shown to cause a loss or marked decrease in the ability of splenocytes to respond to mitogens. These observations have been extended by cell fractionation and reconstitution experiments to show that the mitogen stimulated proliferative capacity of both the CD4+ and CD8+ T cells is affected. Similarly, monocytes which are directly infectable by virus, are functionally defective as antigen presenting cells when added to mitogen stimulated normal T cells. Interestingly, the same T cells which respond poorly in mitogenic assays can be activated by stimulation through the CD3 receptor. In contrast to the defective mitogenic response of T cells, B cell function, as assessed by stimulation through the CD40 ligand in vitro remains intact. There is no evidence for apoptosis in either the T cells or the B cells recovered from the spleens of ASFV infected animals 1-5 days following infection. Although the number of leucocytes which can be recovered from the infected spleen decreases rapidly with progression of the disease, the proportion of the different cell phenotypes remains constant. Thus decreased activity of lymphocytes in lymphoid tissue from ASFV infected animals appears to be directly attributable to infection of the monocytes.     

Protein deficiency induces alterations in the distribution of T-cell subsets in experimental pulmonary tuberculosis

Previous research has suggested that dietary protein deficiency alters resistance to experimental pulmonary tuberculosis, in part, by affecting the distribution and trafficking of antigen-reactive T cells. In this study, guinea pigs were maintained on either a protein-deficient (10% ovalbumin) or control (30% ovalbumin) diet and infected 4 to 6 weeks later with a low dose of virulent Mycobacterium tuberculosis H37Rv by the respiratory route. Monoclonal antibodies directed against the CD4 or CD8 markers on guinea pig lymphocytes were used in a flow cytofluorometric assay to determine the proportion of each subset in the peripheral circulation, spleen, and bronchotracheal lymph nodes at 4 weeks after infection. In uninfected guinea pigs, only the spleen exhibited an effect of diet on T-cell distribution, with small but consistent reductions in the proportions of both CD4 and CD8 T lymphocytes. However, following infection, protein deficiency exerted a profound effect on T-cell distribution. Malnourished, tuberculous guinea pigs harbored only 20 and 60% of the T cells (as a proportion of total lymphoid cells) found in the spleen and blood, respectively, of their well-nourished counterparts. Normal relative proportions of CD4 and CD8 cells were observed, however. In striking contrast, the bronchotracheal lymph nodes of protein-deprived guinea pigs with tuberculosis contained more than twice the numbers of T cells of control guinea pigs, and the normal CD4-to-CD8 ratio was reversed. Peripheral T-cell function, as measured by the delayed hypersensitivity skin test to tuberculin, and antigen-induced lymphoproliferation in vitro were markedly suppressed in protein-malnourished animals. Conversely, purified protein derivative-induced (but not concanavalin A-induced) proliferation was significantly enhanced in cultures of lymph node cells from protein-deprived tuberculous animals. Taken together, these results suggest that immunological abnormalities and loss of antimycobacterial resistance in the lungs of protein-deficient guinea pigs may be explained, in part, by sequestration of antigen-reactive T cells in the lymph nodes draining the site of infection.     

Two new species of tetraphyllidean cestodes in Himantura pacifica (Chondrichthyes: Myliobatiformes: Dasyatididae) from the northwest coast of Costa Rica

The tetrapeptide AcSer-Asp-Lys-Pro (AcSDKP) is a physiological negative regulator of hematopoiesis in mammals. It acts by blocking the cell cycle entry of quiescent stem cells and progenitors. In the present study we report that AcSDKP blocks the proliferation of human as well as chicken lymphocytes. It inhibits by 25 to 40% the polyclonal mitogen- (phytohemagglutinin (PHA), pokeweed mitogen (PWM), or concanavalin A) or mixed lymphocyte reaction-induced proliferation of chicken lymphocytes. A comparable degree of inhibition was observed in human whole blood cultures stimulated by "T" (PHA) or "T and B" (PWM) mitogens. Our results obtained on two phylogenetically distant species show that AcSDKP reduces the lymphocyte proliferation probably by blocking or retarding entry into the cell cycle as previously demonstrated for hematopoietic progenitors and hepatocytes. Therefore, this endogenous, non-species-specific tetrapeptide may be involved in the regulation of immune response.     

Polyclonal immunoglobulin response of thymic, hepatic and splenic lymphocytes from fetal, germ-free and conventionally reared pigs to different B-cell activators

Immunoglobulin (Ig) response to different polyclonal B-cell activators was measured by ELISA in cell culture media of thymocytes, splenocytes and liver cells isolated from pig fetuses, 8-d-old germ-free piglets and conventionally reared pigs. Both in fetal and in postnatal life polyclonally stimulated lymphocytes were found to produce predominantly the IgM isotype; the first IgM formation was detected in 50-d-old fetal liver (gestation in pigs lasts 114 d). Surprisingly, 73-d-old fetal thymic cells were shown to be induced to Ig synthesis and secretion. In contrast to splenocytes of the same age, which secreted exclusively IgM, fetal thymocytes produced IgM, IgG and IgA. Polyclonally stimulated splenic cells as compared with thymic cells started to produce IgA later in fetal ontogeny, whereas the IgG response was not detectable in splenic cell culture media during the whole embryonal development and appeared only after birth. The earliest and the highest Ig stimulation was found after cultivation of lymphocytes with Nocardia delipidated cell mitogen. Interestingly, the moderate stimulatory effect of 65-kDa heat shock protein (Hsp-65) in polyclonal IgM response of fetal splenocytes was observed. We showed that thymic B lymphocytes represent probably the first maturing B cell population detectable in fetal life, which is able to differentiate after polyclonal stimulation into IgM as well as IgA and IgG producing cells.     

The in vitro effect of diethylaminoethyl dextran on stimulation of mouse lymphocytes, and on the mitogenic activity of concanavalin A and lipopolysaccharide

Unpurified lymphocytes from spleen, lymph nodes, thymus, and T and B cell-enriched fractions of spleen cells from NMRI mice as well as lymphocytes of nude mice were cultured and stimulation of the lymphocytes by DEAE-D, Con A and LPS and combinations thereof was measured by incorporation of 3H-thymidine. It was demonstrated that DEAE-D is a rather weak mitogen for mouse lymphocytes, acting apparently on B cells as well as on T cells. Frequency and intensity of lymphocyte stimulation by the T cell mitogen Con A and the B cell mitogen LPS was much better. When combinations of DEAE-D with Con A or LPS, respectively, were used significant enhancement of lymphocyte stimulation occurred, as compared to the mitogens and DEAE-D alone. This enhancement was more prominent with the DEAE-D/Con A combination than with the DEAE-D/LPS combination, and more pronounced with the T cell-rich lymphocyte preparations than with the B cell-rich suspensions. The results are discussed and it is concluded that DEAE-D enhances both T cell and B cell functions, however, T cell functions are favoured. This enhancement is assumed to be mediated by a membrane effect of DEAE-D, and to be responsible for th immunological adjuvant effect of DEAE-D.     

Specific antibody production to a recall or a neoantigen by SCID mice reconstituted with human peripheral blood lymphocytes

To explore the extent of immune reconstitution of SCID mice by human peripheral blood lymphocytes (hu-PBL-SCID mice), we studied the production of immunoglobulin isotypes and specific antibody (Ab) by the engrafted human cells. Human IgG was detectable in 94% of hu-PBL-SCID mice. IgE synthesis by hu-PBL-SCID mice correlated with the IgE levels observed in human donors. All SCID mice receiving PBL obtained from human donors previously immunized with the T-cell-dependent Ag, bacteriophage phi x 174 (phage), produced phage neutralizing antibody. Quantity and quality (Ig isotypes) of phage-specific Ab produced by hu-PBL-SCID mice correlated with that observed in the donor serum. Human B cells alone failed to engraft, and T cells were required for the production of Ig and anti-phage Ab. Phage-specific Ab production occurred without direct Ag exposure of the hu-PBL-SCID mice, suggesting that the specific Ab production was induced directly by polyclonal activation of the engrafted human cells. Intravenous phage injections given 4 wk after cell transfer failed to further increase the anti-phage Ab titer. Phage neutralizing Ab production could not be boosted if spleen cells obtained from hu-PBL-SCID mice were cultured in the presence of Ag. However, hu-PBL-SCID mice produced increased amounts of anti-phage Ab, providing they were injected with phage at the time of cell transfer. Injection of phage at the time of cell transfer, but not 4 wk later, to mice receiving PBL from nonimmunized donors induced production of minute amounts of anti-phage Ab. We conclude that human peripheral blood lymphocytes transferred into SCID mice become maximally stimulated presumably by xenogeneic murine Ag, resulting in polyclonal expansion of the graft and spontaneous production of Ab to Ag the human donor was previously exposed to, and in loss of responses to subsequent Ag exposure. Ab production to neoantigen, however, can be induced and that to recall Ag can be modified if PBL are exposed to Ag at the time of cell transfer.     

Involvement of interleukin 2 receptors in conceptus-derived suppression of T and B cell proliferation in horses

The mechanism by which a horse conceptus-derived immunosuppressive factor (HCS) of M(r) > 100,000 inhibits lymphocyte proliferation was investigated. The factor was obtained from the culture supernatants of 20-day-old horse conceptuses; activity, identified by reduced uptake of [3H]thymidine by mitogen-stimulated lymphocytes, was greatest (P < 0.01) in cultures stimulated by mitogen from pokeweed. HCS also suppressed cell proliferation stimulated by phytohaemagglutinin (P < 0.01), but had no effect on lipopolysaccharide-stimulated cells (P > 0.05). Data from a fluorescence-activated cell sorter indicated that supplementation with HCS reduced the number of T cells in phytohaemagglutinin-stimulated cultures and suppressed proliferation of T and B cells in pokeweed-mitogen-stimulated cultures compared with controls. Cell proliferation was greater (P < 0.01) in cultures supplemented with HCS 24 h after stimulation than in those treated at the start of stimulation, and was even greater (P < 0.01) when cells were treated 48 h after stimulation. The removal of HCS from treated lymphocyte cultures resulted in complete recovery of cell responsiveness, and stimulated proliferation of treated cells did not differ (P > 0.05) from that of control cells. The addition of stimulated equine lymphocyte supernatant to cultures supplemented with HCS did not significantly increase (P > 0.05) cell proliferation in response to pokeweed mitogen. Addition of recombinant human interleukin 2 (rIL-2) to HCS-treated cultures did not alter the suppressive activity of HCS, although cell proliferation was greater in cultures supplemented with rIL-2 than in controls (P < 0.01). HCS inhibition of IL-2 receptor (IL-2R) function was investigated using an IL-2-dependent murine cytolytic T lymphocyte cell line; the fraction of HCS of M(r) > 100,000 had no effect (P > 0.05) on proliferation of IL-2-dependent murine cytolytic T lymphocyte cells induced by rIL-2. Together, these data suggest that HCS suppresses proliferation of T lymphocytes during the early stages of cell activation by inhibiting IL-2R interaction and that this suppression interferes with interactions between T cells and B cells, thereby also indirectly inhibiting proliferation of B cells. The potent immunosuppressive capacity of HCS may be one factor responsible for inhibiting cell-mediated fetal allograft rejection during pregnancy.     

Altered cytokine production and impaired antimycobacterial immunity in protein-malnourished guinea pigs

Protein malnutrition leads to multiple detrimental alterations of host immune responses to mycobacterial infection. In this study, we demonstrated that splenocytes from low-protein (LP) guinea pigs vaccinated 6 weeks previously with attenuated Mycobacterium tuberculosis H37Ra failed to control the accumulation of virulent M. tuberculosis H37Rv in cocultured autologous peritoneal macrophages, despite the fact that they were able to control the accumulation of virulent tubercle bacilli in cocultured syngeneic peritoneal macrophages from normally nourished guinea pigs as successfully as did those from high-protein (HP) counterparts. Vaccine-induced growth control of virulent M. tuberculosis H37Rv in these cocultures appeared to be mediated by CD4 lymphocytes but not CD8 cells. Tuberculin (purified protein derivative [PPD])-induced lymphoproliferation was markedly impaired in vaccinated LP guinea pigs, and the depletion of CD4 lymphocytes significantly decreased lymphocyte proliferation whereas CD8 cell depletion did not. Protein malnutrition also impaired the abilities of cells from vaccinated LP guinea pigs to produce cytokines, including interferon, tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta), in response to PPD, despite the demonstration of higher serum levels of TNF-alpha and TGF-beta after an intravenous injection of PPD into LP guinea pigs. In contrast, peritoneal macrophages from protein-malnourished guinea pigs produced a higher level of TGF-beta 4 days after infection in vitro with M. tuberculosis H37Rv than did those from protein adequate controls. These results suggest that dietary protein malnutrition impairs vaccine-induced resistance to M. tuberculosis, in part, by altering the cytokine profile to favor macrophage deactivation.     

Urinary excretion of meperidine and its metabolites

The urine of male and female mice, rats, guinea pigs, rabbits, cats, and dogs, given meperidine hydrochloride, 20--40 mg/kg ip, was analyzed by GLC for meperidine, normeperidine, p-hydroxymeperidine, and total (free and conjugated) meperidinic and normeperidinic acids. More than 90% of the excreted drugs was found in the 24-hr urine. Meperidine was observed in the urine of mice, rats, guinea pigs, and cats, but only a trace amount was observed in the urine of rabbits and dogs. Normeperidine, p-hydroxymeperidine (except in the mice), and total meperidinic and normeperidinic acids were observed in all species. All of the species studied have the capacity to N-demethylate meperidine to normeperidine and to hydrolyze meperidine and normeperidine to their respective acids. The male has a higher N-demethylating activity that the female with the exception of mice. Ester hydrolysis is a major metabolic pathway for meperidine metabolism.     

Purification, and biochemical and biological characterization of an immunosuppressive and lymphocyte mitogenic protein secreted by Streptococcus sobrinus

An immunosuppressive/mitogenic (ISM) protein was purified from the supernatants of cultures of Streptococcus sobrinus with an isoelectric point of 4.75 and a relative molecular mass of 38 kDa (p38). Treatment of C57BL/6 mice with p38 induced an increase in the numbers of non-specific splenic Ig-secreting plaque-forming cells (PFC) with peak responses on day 3 for IgM-secreting PFC and on day 5 for IgG-secreting PFC, with an isotype pattern consisting predominantly of IgG2a and IgG2b. This increase was accompanied by a lymphocyte blastogenic response of both T and B lymphocytes. The in vitro effects of p38 on pure B, T and total splenic lymphocytes indicated that this ISM protein was primarily a B cell mitogen, being T cells activated subsequently by the generation of B blasts. Suppression of the murine primary immune response against sheep red blood cells was observed in C57BL/6 mice treated 4 days before with p38. The amino acid sequence of the N-terminus of p38 has a significant similarity with several enolases, particularly with rabbit enolase. However, the biological effects ascribed to p38 have not been detected after in vivo treatment with that enolase. The immunosuppressive effect of p38 was abrogated by depletion of IL-10 but not of IL-4. In agreement with this observation IL-10 was the only cytokine detected in serum of C57BL/6 mice after p38 treatment and the peak of serum levels was observed as soon as 2 h after treatment.     

Involvement of IFN-gamma and transforming growth factor-beta in graft-vs-host reaction-associated immunosuppression

The mechanisms of the suppressive activity of spleen cells from mice undergoing a graft-vs-host reaction (GVH) to non-H-2 histocompatibility Ag were investigated. In our model GVH is induced by injecting bone marrow and spleen cells from B10.D2 (H-2d Mlsb) donors into lethally irradiated (DBA/2 x B10.D2)F1 (H-2d/d Mlsa/b) recipients that differ only with regard to non-H-2 Ag. GVH spleen cells inhibit the mitogenic responses to Con A and LPS, as well as the anti-bromelain-treated mouse RBC (Br-MRBC) antibody response. This suppression was nonspecific and non-H-2-restricted and was not modified after treatment with anti-Thy-1 plus C. Conversely it was abrogated after treatment with L-leucyl methyl ester. These features permitted the identification of non-T cell, L-leucyl methyl ester-sensitive, cells involved in this type of suppression. The suppression mediated by GVH spleen cells was linked to the activity of IFN-gamma and transforming growth factor-beta 1 (TGF-beta 1) (TGF-beta 1 was found to be synthesized by GVH spleen T cells). mAb to IFN-gamma abrogated the suppression of the mitogenic response to Con A and the anti-Br-MRBC response and slightly reversed the suppression of the mitogenic response to LPS. Anti-TGF-beta 1 antibody partially abrogated the suppression of the mitogenic response to LPS and totally abrogated that of the anti-Br-MRBC response but left unmodified the suppression of the mitogenic response to Con A. These results are discussed within the framework of the mechanisms underlying the immunosuppression associated with GVH.     

Drug-induced xerostomia

Immunity relies on the circulation of lymphocytes through many different tissues including blood vessels, lymphatic channels, and lymphoid organs. The ability of lymphocytes to traverse the interstitium in both nonlymphoid and lymphoid tissues can be determined in vitro by assaying their capacity to locomote through Type I collagen. In an attempt to characterize potential causes of microgravity-induced immunosuppression, we investigated the effects of simulated microgravity on human lymphocyte function in vitro using a specialized rotating-wall vessel culture system developed at the Johnson Space Center. This very low shear culture system randomizes gravitational vectors and provides an in vitro approximation of microgravity. In the randomized gravity of the rotating-wall vessel culture system, peripheral blood lymphocytes did not locomote through Type I collagen, whereas static cultures supported normal movement. Although cells remained viable during the entire culture period, peripheral blood lymphocytes transferred to unit gravity (static culture) after 6 h in the rotating-wall vessel culture system were slow to recover and locomote into collagen matrix. After 72 h in the rotating-wall vessel culture system and an additional 72 h in static culture, peripheral blood lymphocytes did not recover their ability to locomote. Loss of locomotory activity in rotating-wall vessel cultures appears to be related to changes in the activation state of the lymphocytes and the expression of adhesion molecules. Culture in the rotating-wall vessel system blunted the ability of peripheral blood lymphocytes to respond to polyclonal activation with phytohemagglutinin. Locomotory response remained intact when peripheral blood lymphocytes were activated by anti-CD3 antibody and interleukin-2 prior to introduction into the rotating-wall vessel culture system. Thus, in addition to the systemic stress factors that may affect immunity, isolated lymphocytes respond to gravitational changes by ceasing locomotion through model interstitium. These in vitro investigations suggest that microgravity induces non-stress-related changes in cell function that may be critical to immunity. Preliminary analysis of locomotion in true microgravity revealed a substantial inhibition of cellular movement in Type I collagen. Thus, the rotating-wall vessel culture system provides a model for analyzing the microgravity-induced inhibition of lymphocyte locomotion and the investigation of the mechanisms related to lymphocyte movement.